The histone methyltransferase EZH2 is overexpressed in glioblastoma multiforme (GBM) tumors as well as glioma stem cells (GSCs), and functions as an oncogene. It is known to function primarily as an epigenetic repressor of gene expression by methylating histone H3K27. EZH2 is the catalytic component of the polycomb repressive complex PRC2, which has important roles in cancer, pluripotency and other fundamental cellular processes. Recent work has shown that PRC2 also associates with long non-coding RNAs (lncRNAs), as well as intronic and promoter-associated non-coding RNAs. The specificity of PRC2 association with non-coding RNAs and the role of these associated RNAs in mediating the function of PRC2 is under active investigation. We have discovered that in GBM cancer cells, EZH2 binds to short RNAs that are derived from the exons of protein coding genes. This exonic RNA association of EZH2 has not been noted before, and the significance of this binding with regard to regulating EZH2 function is unknown. However, many of these target genes have known functions in GBM biology. Importantly, we have also found that loss of EZH2 reduces the expression of several of these target RNAs, indicating that EZH2 can function as an activator of gene expression by a potentially novel mechanism. We hypothesize that in GBM, EZH2/PRC2 could have a role in regulating gene expression and/or splicing that is independent of its established roles in histone methylation, and mediated instead by its association with exonic RNAs. This novel role for PRC2 will be tested in GBM cancer cells and glioma stem cells (GSCs) in this project. In Aim 1, we will use RNA immunoprecipitation and RNA crosslinking based sequencing approaches (RIP-seq and iCLIP-seq) to identify all EZH2-associated exonic RNAs in GBM cancer cells and GSCs . We will determine whether other PRC2 components also associate with exonic RNAs, indicative of the PRC2 complex as a whole functioning in this novel manner. We will also establish whether EZH2 or PRC2 is recruited to genomic loci at the regions where it associates with exonic RNAs, which would be indicative of a co-transcriptional mechanism. In Aim 2 we will establish whether loss of EZH2 affects gene expression levels and alternative splicing of the exonic targets that it binds to, and test if ectopic expression of these RNAs can direct EZH2 recruitment to specific chromosomal sites. These studies point to a novel mechanism of function for EZH2 or the PRC2 complex in GBM, and stand to reveal new ways in which it can impact the biology of GBM and thus offer new avenues for therapeutic intervention.